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UNIVERSITI PUTRA MALAYSIA
MODIFYING THE RESPONSE OF MALE BROILER CHICKENS TO HEAT STRESS THROUGH EARLY AGE FEED RESTRICTION AND
THERMAL CONDITIONING
LIEW PIT KANG
FP 2002 27
MODIFYING THE RESPONSE OF MALE BROILER CHICKENS TO HEAT STRESS THROUGH EARLY AGE FEED RESTRICTION AND THERMAL
CONDITIONING
By
LIEW PIT KANG
Thesis Submitted to the School of Graduate Studies, University Putra Malaysia in Fulfilment of the Requirements for tbe Degree of Master of Science
September 2002
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science
MODIFYING THE RESPONSE OF MALE BROILER CHICKENS TO HEAT STRESS THROUGH EARLY AGE FEED RESTRICTION AND
THERMAL CONDITIONING
By
LIEW PIT KANG
September 2002
Chairman: Associate Professor Dr. Zulkifli Idrus, Ph.D.
Faculty: Agriculture
Two experiments were conducted to investigate the effects of early age feed
restriction and heat conditioning on tolerance to acute and chronic heat stress in
male broiler chickens. In both experiments, equal numbers of chickens were
subjected to (i) 60% feed restriction on day 4, 5, and 6 (FR), (ii) exposure to
36±IOC and 50-60% relative humidity for 1 hour from day 1 to 21 (HT), (iii)
60% feed restriction on day 4, 5, and 6 and exposure to 36±loC and 50-60%
relative humidity for 1 hour from day 1 to 21 (FRHT). (iv) ad libitum feeding
and no heat treatment (control). In experiment I, on day 35, all birds were
exposed to 39±loC for 6 hours and 50% relative humidity. Subjecting chicks to
FR, HT and FRHT reduced HLR response to the heat challenge. Following heat
exposure, the FR and FRHT chick had greater heat shock protein (hsp) 70
density than those of controls. The hsp 70 response of HT birds was not
significantly different from the other three groups. The FRHT birds were more
hyperthermic than controls during heat challenge. In expe� II. from day 36
ii
- 50, all birds were exposed to 38±l oC and 80 % relative humidity for 2
hours/day. One day following heat exposure (day 37), all birds were
administrated intranasally with infectious bursal disease (IBD) vaccine virus.
The dosage used was l Ox of the recommended level. Subjecting chicks to FRHT
improved relative weight gain and resistance to IBD infection and reduced HLR
in response to the heat treatment as compared with the control birds. Although
there is evidence that FR and HT can improve heat tolerance, the FRHT
combination may further enhance the ability of birds to withstand chronic heat
stress. The acquired improved heat tolerance resulting from FRHT, FR, and HT
could be attributed to enhanced hsp 70 response. The trend of hsp 70 response
correlated well with IBD lesion scores, suggesting hsp 70 may play a role in
resistance against viral infection. Based on experiment I and II, it can be
concluded that the present findings confirmed earlier studies that FR is effective
in alleviating the adverse effects of heat stress. Subjecting birds to FRHT can
further improve tolerance to chronic but not acute heat stress.
III
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Master Sains
MENGUBAH GERAKBALAS AYAM PEDAGINGJANTAN TERHADAP TEGASAN HABA MELALUI SEKATAN MAKANAN AWAL HAYAT
DAN PENYESUAIAN THERMA
Oleh
LIEW PIT KANG
September 2002
Pengerusi: Professor Madya Dr. Zulkifli Idrus, Ph.D.
Fakulti: Pertanian
Dua eksperimen telah dijalankan untuk menyiasat kesan sekatan makanan awal
hayat dan penyesuaian thenna terhadap tegasan haba yang akut dan kronik dalam
ayam pedaging jantan. Pada kedua-dua eksperimen, bilangan ayam yang sama
telah didedahkan kepada (i) 60% sekatan makanan pada hari 4, 5 dan 6 (FR), (ii)
36±lOC dan 50-60% kelembapan relatif selama 1 jam dari hari 1 ke 21 (Hn, (iii)
60% sekatan makanan pada hari 4, 5 dan 6 dan 36±loC dan 50-60% kelembapan
relatif selama 1 jam dari hari 1 ke 21 (FRHn, (iv) pemberian makanan secara ad
libitum dan tidak ada perlakuan haba (kawalan). Dalam eksperimen I, pada hari
ke-35, semua ayam telah didedahkan kepada tegasan haba setinggi 39±I°C
selama 6 jam dengan 50% kelembapan relatif. Pendedahan ayam kepada FR, HT
dan FRHT mengurangkan gerakbalas nisbah heterofilllimfosit (HLR) terhadap
tegasan haba. Berikutan tegasan haba, ayam FR dan FRHT mempunyai
ketumpatan protein kejutan haba (hsp) 70 yang lebih tinggi daripada ayam
kawalan. Penghasilan hsp 70 oleh ayam HT adalah sama dengan ketiga-tiga
iv
kumpulan yang lain. Suhu badan ayam FRHT adalah lebih tinggi daripada ayam
kawalan semasa tegasan haba. Dalam eksperimen II, dari hari 36-50, semua
ayam telah didedahkan kepada 36±1 °C dan 80% kelembapan relatif selama 2
jam/hari. Sehari selapas tegasan haba (hari 37), semua ayam dicabaran dengan
menggunakan virus vaksin penyakit berjangkit bursal (IBD). Pendedahan ayam
kepada FRHT meningkatkan penambahan berat badan dan ketahanan terhadap
cabaran IBD, dan mengurangkan peningkatan HLR akibat tegasan haba
berbanding dengan ayam kawalan. Walaupun ada bukti menunjukkan bahawa
FR dan HT boleh meningkatkan tahanan terhadap tegasan haba, kombinasi
FRHT boleh meningkatkan lagi keupayan ayam untuk menahan suhu
persekitaran yang tinggi. Peningkatan toleransi terhadap suhu yang diperolehi
daripada FRHT, FR, and HT mungkin ialah hasil daripada peningkatan
gerakbalas hsp 70. Trend untuk penghasilan hsp 70 berkait rapat dengan skor lesi
IBD,�mencadangkan bahawa hsp 70 mungkin terlibat dalam ketahanan terhadap
jangkitan virus. Berdasarkan kepada eksperimen I dan II, kesimpulan yang boleh
dibuat ialah keputusan eksperimen megesahkan kerja-kerja yang lebih awal iaitu
FR adalah berkesan untuk meringankan kesan-kesan buruk akibat tegasan haba.
Pendedahan ayam kepada FRHT meningkatkan lagi toleransi terhadap tegasan
haba kronik tetapi bukan akut.
v
ACKNOWLEDGEMENTS
First and foremost, I would like to express my sincerest thanks and
gratitude to my supervisor Dr. Zulkifli Idrus for his guidance, advice, patience,
and support throughout the course of this study. Gratitude is also extended to Dr.
Mohd. Hair Bejo, Dr. Abdul Rahman Omar and Dr. Daud Ahmad Israf Ali for
their advice and invaluable help throughout the course of the present study.
Heartfelt appreciation also goes to Dr. Ho Yin Wan, Dr. Abdul Rani
Bahaman and Dr. Loh Teck Chwen for granting permission to use equipment and
facilities in their laboratory and precious support.
The help from staff working at poultry research farm of UPM, Mr.
Ponnusamy, Mr. Mazlan, Mr. Islahuddin, and Mr. Khairul and Mr. Nagaya from
Institute of Bioscience during the experiments are gratefully acknowledge. I wish
to thanks Mr. Ibrahim Mohsin, Mr. Saparin and Kak Sapiah for their help and
technical skills.
Many thanks are also extended to Han Chern, Dr. Phong, Li Kim, Lih
Ling, Siaw Wee, May Ling, Bee Koon, Chin Chin, Kala, Latifah, Mdm. Haw,
and Ganqiu for their friendship and invaluable assistance.
VI
To my oid friends, Albert Lee, Maximilian and Hooi Ching, thank you
for your friendship, support and advice. To my dog, Popeye thanks for your
companionship, I will always remember the wonderful memories of days past.
Finally, I deeply indebted to my parents, brother, sisters and Hwei San for
their endless support, encouragement, patience and sacrifices during my study in
UPM campus.
Vll
I certify that an Examination Committee met on 20th September 2002 to conduct the fmal examination of Liew Pit Kang on his Master thesis entitled "Modifying the Response of Male Broiler Chickens to Heat Stress Through Early Early Age Feed Restriction and Thermal Conditioning" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1 980 and Universiti Pertanian Malaysia Regulations (Higher Degree) 1 981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follow:
LIANG JUAN BOO, Ph.D. Associate Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Chairman)
ZULKIFLI IDRUS, Ph.D. Associate Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Member)
MOHD HAIR BEJO, Ph.D. Associate Professor, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Member)
DAUD AHMAD ISRAF AL�, PhD. Associate Professor, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, (Member)
ABDUL RAHMAN OMAR, Ph.D. Lecturer, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Member)
CSHi'MSHER MOHAMAD RAMADILI, Ph.D. ProfessorlDeputy Dean, School of Graduate Studies, Universiti Putra Malaysia
Date: I. 8 0(; I 2002
viii
This thesis submitted to the senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirements for the degree of Master of Science. The members of the Supervisory Committee are as follow:
ZULKIFLI IDRUS, Ph.D. Associate Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Chairman)
MOHD HAIR BEJO, Ph.D. Associate Professor, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Member)
DAUD AHMAD ISRAF ALI, PhD. Associate Professor, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, (Member) .
ABDUL RAHMAN OMAR, Ph.D. Lecturer, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Member)
AINI IDERIS, Ph.D. ProfessorlDean, School of Graduate Studies, Universiti Putni Malaysia.
Date:
ix
DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that this thesis has not been previously or concurrently submitted for any other degree at UPM or any institutions.
LIEW PIT KANG
Date: "'70 f q 1290'2-
x
TABLE OF CONTENTS
PAGE
lllJ�1r�<:1r.................................................................. ii AB�1r�..................................................................... iv ACKN'OWLEDGEMENTS...... ••• ••••. .•........ ..... •••. •.•••. •••....•.. vi APPROVAL SHEETS... .•..•.•....••••.••..•.•.•.. ••••.• ••..•..• ..••.•••••• viii DECLARATION FORM.................................................... x �IST ()lr TAB�ES........................................................... xiii �I�T ()� ��)\��............................................................ xi" �IST (»)r ABIJ��1fI()�S.............................................. �
CHAPTER
I INTRODUCTION................................................... 1
II LITE�T�REVIEW......................................... 6 Introduction and Theory of Stress...... . . . . . . .................. .... 6 Prior Experience and Response to A Subsequent Stressor.... 8 Feed Restriction................................ ..................... 11 Heat Stress............... . .............. ... . .. . . ...... ....... ........ 13 Heat Stress and Poultry Production. .. . . . . . . . . . . ... . . ... . . . . .. . . . 21 Heat Shock Protein.......... . . . . . . . . . . . ... .. ..... ........ .... ... . ... 24
III EFFECTS OF EARLY AGE FEED �STRICTION AND THERMAL CONDITIONING ON HETEROPHIULYMPHOCYTE �TIOS, HEAT SHOCK PRO�IN 70 EXP�SSION AND BODY TEMPE�T� OF ACUTE HEAT-STRESSED �E Bll()ILEll C1B[[C�NS................................. 31
Introduction.. . . . . . ... ..... ... .. . .. .... ....... ..... ...... ...... ... .... 31 Materials and Methods.. . . . . . . . . . . ... . .. . .. ... ...... ......... .. ..... 34
Birds, Husbandry, Experimental Procedure and Traits Measured.. . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . 34 Acute Heat Challenge and Traits Measured... . . .. . . . . .. 35 Determination of hsp 70 Concentration..... . . . . . . . . . . ... 36
Determination of Protein Concentration. . . . . ... 36 Protein Separation.. . . . . . . . . . . . . .... . . . . ..... . . . . ... 38 Immunoblotting of hsp 70........................ 40 Molecular Weight Determination and Quantification of hsp 70...................... 42
Statistical Analyses. . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . ..... 43 Results. ... . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . .. . . ..... 44 Discussion.. . . . .. . . . . . . . . . . ... . . . . .. . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . .. 50 Conclusion.. . .... . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . ....... . . . . . . ... 55
Xl
IV EFFECTS OF EARLY AGE FEED RESTRICTION AND THERMAL CONDITIONING ON HEAT SHOCK PROTEIN 70 RESPONSE, DISEASE RESISTANCE AND TOLERANCE TO CHRONIC HEAT STRESS IN MALE BROILER CHICKENS ............................. ' " 56
Introduction.... . . . . . . . . . . . .. . ..... .... ...... ... ... . .. ... .... ....... ... 56 Materials and Methods... .. . .. . ...... ...... .. . ............ .......... 59
Birds, Husbandry, Experimental Procedure and Traits Measured.... . . . . . . . . . . . . . . . . . . . . ..... ... . . . . . . . . 59 Heat Challenge and Traits Measured..... . . . . . ..... . ..... 60 Determination of hsp 70 Concentration ... . . ,. ..... . ..... 62 Determination of Infectious Bursal Disease and Newcastle Disease Vaccination-Specific Antibody Titres .. . . .... ... . . . . . .... .. .. ... " . .. .. .. . . . .. . .. ... 62 Lesion Scoring of Bursa of Fabricius. ..... . . . . . . . . . . . ..... 63
Sample Preparation...... . . . .. . ........... ........... 63 Histopathology. . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . .. .. 64
Statistical Analyses... . . . . . . .. . ...... . .. ................. .... 65 Results.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... . . . . . . . 66 Discussion. . . . ..... . .. .. . . ..... ... ..... . . .. . . . .. . .. . .. . . ...... . .. . . . .... 81 Conclusion . . . . .. . . .... .. . . .. . . . . . . . . . . . . . .... ...... , " . . . . . . . . . . . . . .... 86
V GENERAL DISCUSSION AND CONCLUSION............... 87
BIBIOGRAPHY...................................................... 91
APPENDICES........................................................ I I I
VITA................................................................... 125
Xll
Table
1
2
3
4
5
6
LIST OF TABLES
Mean heterophilllymphocyte ratios (±SEM) where stage of heat exposure by treatment interactions were
. ·fi ' Slgm lcant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . .... .
Mean heat shock protein 70 densities (±SEM) where stage of heat exposure by treatment interactions were significant . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .
Mean rectal temperatures (±SEM) where stage of heat exposure by treatment interactions were significant . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . ..... .
Mean body weight (g) (±SEM) of broiler chickens by early age treatment at various ages . . . . . . . . . . . . . . . . . . . . ...... . . .
Mean relative weight gains (±SEM) {(BW day 49-BW day 35)/BW day 35} of broiler chickens by treatment during heat exposure .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mean heterophilllymphocyte ratios (±SEM) where age by treatment interactions were significant .. . . . . . . . . . . . . . . . .. . . . .
7 Mean antibody titre (±SEM) against Newcastle disease (NO) virus and infectious bursal disease (IBD) virus challenge by
Page
46
47
49
69
70
71
age and treatment ...................................................... 72
8 Mortality rate (%) of broiler chickens by treatment during heat exposure. . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . 73
9 Mean heat shock protein 70 densities (±SEM) where age by treatment interactions were significant. . . . . . . . . . . . .. . . . . . . .. .. 74
10 Mean bursal to body weight ratios (±SEM) by age and treatment . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .... 77
11 Mean bursal lesion scores (±SEM) where age and treatment interactions were significant.. . . . . . . . . . . . . . . . .... . .. 78
xiii
LIST OF PLATES Plate Page
1 PVDF membrane representation of hsp 70 synthesis in the brain of chickens prior to heat exposure (0 hour) by early age treatment. Molecular weight standard (GibcoBRL) and their molecular size (kDa)are indicated . . . . . . . . . . . . . . .... . . 48
2 PVDF membrane representation of hsp 70 synthesis in the brain of chickens following 3 hours of heat exposure by early age treatment. Molecular weight standard (GibcoBRL) and their molecular size (kDa) are indicated . . . . . . . . . . . . . . . . . . 48
3 PVDF membrane representation of hsp 70 synthesis in the brain of chickens following 6 hours of heat exposure by early age treatment. Molecular weight standard (GibcoBRL) and their molecular size (kDa) are indicated . . . . . . . . . . . . . . . . . . 48
4 PVDF membrane representation of hsp 70 synthesis in the brain of chickens prior to heat exposure by early age treatment. Molecular weight standard (GibcoBRL) and their molecular size (kDa) are indicated . . . . . . .. . . . . . . . . . . . . . . . . 75
5 PVDF membrane representation of hsp 70 synthesis in the brain of chickens on day 39. Molecular weight standard (GibcoBRL) and their molecular size (kDa) are indicated . . . . 75
6 PVDF membrane representation of hsp 70 synthesis in the brain of chickens on day 41. Molecular weight standard (GibcoBRL) and their molecular size (kDa) are indicated ... . 75
7 PVDF membrane representation of hsp 70 synthesis in the brain of chickens on day 44. Molecular weight standard (GibcoBRL) and their molecular size (kDa) are indicated ... . 76
8 PVDF membrane representation of hsp 70 synthesis in the brain of chickens on day 51. Molecular weight standard (GibcoBRL) and their molecular size (kDa) are indicated . . . . 76
9 Bursa of Fabricius of HT chicken prior to IBD challenge. Lesion score of O. HE, lOX . ..................................... 79
10 Bursa of Fabricius of HT chicken at 51 days of age. Lesion score of 3. HE, lOX . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 79
11 Bursa of Fabricius of HT chicken at 44 days of age. Lesion score of 5. HE, lOX . ...................................... 80
XIV
ACTH
ANOVA
APS
ATP
ATPase
BCIPINBT
BSA
CP
CRF
ddH20
ELISA
DNA
EDTA
FCR
FR
FRHT
g
GaINAcT
GAS
GLM
H
HE
LIST OF ABBREVIATIONS
Adrenocorticotrophic Hormone
Analysis of Variance
Ammonium Persulfate
Adenosine Triphosphate
Adenosine Triphosphatase
5-Bromo-4-Chloro-3-Indolyl-PhasphatelNitroblue Tetrazolium
Bovine Serum Albumin
Crude Protein
Corticotrophin-Releasing Factor
Deionised Distilled Water
Enzyme-linked Immunosorbent Assays
Deoxyribonucleic Acid
Ethylene-Diamine-Tetraacetic Acid
Feed Conversion Ratio
60% Feed Restriction on day 4, 5, and 6
Combination of FR and HT
Gravity
N -acetylgalactosaminyl transferase
General Adaptation Syndrome
General Linear Models
Heterophil
Haematoxylin and Eosin
xv
HLR Heterophil Lymphocyte Ratio
HP A Hypothalamic-Pituitary-Adrenal
hsp Heat Shock Protein
HT Exposed to 36±I°C and 50-60% relative humidity for 1 hour from
day 1 to 21
IBD Infectious Bursal Disease
IDV Integrated Density Value
IGF-I Plasma Insulin-like Growth Factor-I
KCal KiloCalorie
kDa KiloDalton
kg Kilogram
L Lymphocyte
ME Metabolisable Energy
J.1 Micron
J.1g Microgram
J.11 Microlitre
mA Mili-Ampere
ml Millilitre
mm Millimetre
mRNA Messenger Ribonucleic Acid
nm Nanometer
NCM Negative Control Mean
ND Newcastle Disease
PCM Positive Control Mean
XVI
PVDF
rpm
RH
Rr
SAS
SDS
Polyvinylidene Diflouride
Revolutions Per Minute
Relative Humidity
Relative Mobility
Statistical Analysis System
Sodium Dodecyl Sulphate
SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
SIP Sample to Positive Ratio
T3
T ..
TEMED
TTBS
V
Wk
3,3' ,S-Triiodothyronine
Tetraiodothyronine
N, N, N', N'-Tetramethyleethylenediamine
Tween-20-Tris-Buffered saline
Volt
Week(s)
XVll
CHAPTER I
INTRODUCTION
Climatic conditions have always been critical in poultry production. High
temperatures in combination with high humidity create an undesirable
environment that may cause extreme thermal stress in poultry. This harsh
environment is not uncommon in tropical countries where the temperature and
relative humidity are high. Although birds can survive within wide range of
temperature, temperature above the comfort zone will certainly compromise the
actual genetic potential of the birds. This is mainly due to elicitation ·of abnormal
physiological, hormonal, and behavioural responses under the thermal stress
condition. Furthermore, intense selection for high growing rate, increase stocking
density, and green house effects definitely will direct or indirectly exacerbate the
thermal stress conditions. It is a tremendous challenge to poultry producers to
minimize the losses attributed to the hot and humid conditions.
Extensive studies have been done in the past decades to combat the
adverse effects of thermal stress and to improve the performance of poultry
under such conditions. As it is expensive to cool animal buildings, several
methods have been examined to alleviate the effects of high environment
temperatures on poultry performance and well being. These approaches included
genetic manipulation (Bohren et al., 1982; EI-Gendy and Washburn, 1989),
dietary supplementation of anti-stress agents such as vitamins and electrolytes
(Pardue and Thaxton, 1 982; 1 986; Kafri and Cherry, 1 984; Pardue et al., 1 985a;
b; Gross, 1 988; Ferket and Qureshi, 1 992; Koelkebeck et al., 1 993; Ait
Boulahsen et al., 1 995; Mckee and Harrison, 1 995; Bollengier-Lee, et al., 1 998;
Chen and Balnave, 2001 ; Puthpongsiripom et al., 2001 ), manipulation of poultry
diet such as provision of high fat and low protein diet (Ramlah and Sarinah,
1 992), and short term fasting (McCormick et al., 1 980a; b; c; Preston, 1 987;
Zulkifli and Fauzi, 1 996).
Habituation through acclimatization is essential and vital in helping an
animal to survive in an environment in which it has to live. Early age stressful
experiences may have long-lasting beneficial effects in modifying tolerance to
thermal stress in poultry. Work by Arjona et al. (1 988), and Yahav and Hurwitz
(1 996), demonstrated that birds preconditioned to heat stress early in life were
better able to withstand high ambient temperature later in life. Acclimation to
heat stress may also occur by preconditioning to non-temperature stressor.
Bowen and Washburn (1 984) acclimated chickens at 21 to 24 days of age by
repeated handling. Work by Zulkifli et al. (1 994a; b; 2000) reported that neonatal
feed restriction, as indicated by growth, disease resistance, humoral immunity,
survivability and leucocytic count, enhanced heat tolerance at juvenile stage. At a
molecular level, thermotolerance by exposure to controlled thermal stress or mild
feed restriction early in life has been associated with enhanced heat shock protein
(hsp) 70 expression (Wang and Edens, 1 993; 1 994; 1 998; Zulkifli et al., 2002).
2
It is well documented that when prokaryotic and eukaryotic cells are
exposed to a variety of physiological stressors, the synthesis of most proteins is
suppressed but a small number of proteins known as hsp are rapidly synthesized
(Tissieres, 1 974; Ashburner and Bonner, 1 979; Lindquist, 1 986). The synthesis
of hsp is temperature- and time-dependent. The amount of hsp synthesized
depends upon the severity of the hyperthermia or non-thermal stressor. The
synthesis rate accelerates dramatically when the incubation temperature was
raised to a level above body temperature and tends to diminish after the
temperature reaches critical level at 45°C (Wang and Eden, 1993). Generally,
hsp can be classified into 3 families by molecular weight and functions. These
proteins have molecular weight of approximately 90, 70 and 27 kiloDalton (kDa)
and referred to as hsp 90, hsp 70 and hsp 27, respectively (Itoh and Tashima,
1 991 , Lindquist, 1 986). There is another group of hsp called ubiquitin. The
molecular weight is between 7 to 8 kDa. Ubiquitin has recently found to be heat
inducible in both yeast and chickens (Lindquist, 1 986). During thermal stress,
heat shock factor is interacts with heat shock elements, which is a conserved
DNA sequence, to produce hsp. It also binds to other regions of genome,
suppressing translation and transcription of other genes. Hsp produced will bind
to heat sensitive site, for example nucleus, during thermal stress. Besides, it may
bind to cellular protein that have been denatured by the heat and prevent their
catastrophic precipitation. Of the many hsp, hsp 70 is one of the most studied.
Hsp 70 is evolutionarily conserved and appears to correlate best with heat
resistance, either transient or permanent (Li and Werb, 1 982, Lindquist, 1986,
Craig and Gross, 1 991). Hsp 70 acts as a molecular chaperone by binding to
3
newly synthesized proteins and prevent them from aggregating and precipitating
before they are properly folded, transported and incorporated into complexes of
organelles during stress (Yahav and Hurwitz, 1996). There is strong evidence
that a causal relationship exists between the enhanced synthesis of hsp and cell
survival under specific stress (Gloria, 1983). Barbe et al. (1988) reported that hsp
is to protect organisms from detrimental effects of heating. In the complexity of a
homeotherm animal, there is a positive correlation between thermotolerance and
concentration of hsp 70 (Givisiez et al., 1999).
Mother nature is unpredictable. Scientific ways to ameliorate the adverse
effects of heat stress must have long-lasting impact that protects the birds from
undesirable external environment changes. Short term fasting prior to thermal
stress is less practical under field condition mainly due to unpredictable weather.
Continue dietary supplementation of anti-stress agents such as vitamins and
electrolytes is costly. In the search of other solutions, habituation gives an
alternative way. Although early age heat conditioning (Wang and Edens, 1998)
and neonatal feed restriction (Zulkifli et al., 1994a, b; 2000, 2002) can improve
the production of hsp 70, it is unknown whether the combination of these two
techniques will further improve heat tolerance in broiler chickens. To the best of
my knowledge, the only documented study on the effect of early age heat
conditioning and fasting was on heat tolerance in broiler by Yahav and Plavnik
(1999). They concluded that thermal conditioning has advantages over feed
restriction and the combination of both. On a cautionary note, however, the feed
restriction regimen practiced by the authors was more severe than those of
4
Zulkifli et aZ. (2000). Interestingly, Zulkifli et aZ.· (2000) found that the
magnitude of stress perceived early in life was critical in determining the levels
of tolerance to a subsequent stressor in broiler chickens. Optimum level of stress
early in life may result beneficial long-lasting physiological alterations.
Most research on hsp in poultry has emphasized its association with
performance, body temperature and survivability (Givisiez et aZ., 1999 , Wang
and Eden, 1993, Yahav et aZ., 1997). There is a paucity of information on the
relationship among hsp activity and disease resistance under stressed conditions
in poultry. Zulkifli et aZ. (l994a) reported early feed restriction improved
resistance to marble spleen disease in heat stress birds. Works by Zulkifli et aZ.
( 2000, 200 2) suggest a possible positive relationship between ability to produce
hsp 70 and viral disease resistance in poultry.
The objectives of the studies were
1 . To evaluate the effects of early age feed restriction, thermal
conditioning and their combinations on heat stress in male broiler
chickens.
2. To determine hsp 70 response in heat-stressed male boiler chickens.
3. To ascertain the relationship between hsp 70 and resistance to
infectious bursal disease in heat-stressed broiler chickens.
5
CHAPTER II
LITERATURE REVIEW
Introduction and Theory of Stress
Stress is very abstract. Just like we say , A flower is beautiful', but
'beauty' itself is abstract. We will never see 'stress' but we can always feel it
because 'stress' cannot and should not be avoided. The definition of stress is
imprecise and very subjective during the early stage of research. Even until now,
the definition of stress in dictionary still unclear. According to The Concise
Oxford Dictionary ( 1995) stress is 'a demand on physical or mental energy, and
distress is cause by stress'. Bailliere's Comprehensive Veterinary Dictionary
( 1988) defined stress as 'the sum of the biological reactions to any adverse
stimulus, physical, mental or emotional, internal or external, that tends to disturb
the homeostasis of an organism, and should these reaction be inappropriate, they
may led to disease states'. Scientifically, a precise definition to describe the
physiological responses by unfolding the mechanism of stress underneath is
essential.
During the first half of the twentieth century, scientists had gradually
formulated the concept of stress and elucidated the overall response mechanisms.
Walter B. Cannon, a Harvard physiologist who called the power to maintain
6
constancy in livings homeostasis discovered the 'fight and flight response'. On
the other hand, Hans A. Selye, the grandmaster of stress research who defined
'stress' as the non-specific response of the body to any demand, proposed the
'General Adaptation Syndrome' (GAS). 'Fight and flight response' is a
coordinated result of increased secretion of neurogenic amines (catecholamines),
notably, adrenaline by adrenal medulla and noradrenaline by the postganglionic
sympathetic nerve ending. It is a short-term response that creates the optimum
situation for the survival of the animal by fighting the adversary or fleeing from
it. Whereas, the GAS is a longer-term adjustment of the animal to environment
change. It was first published in Nature in 1936 and subsequent description of
the details in 1937. Generally, a stress agen� called stressor, will elicit GAS as a
result of disturbances of the milieu interieur of the body. It involves the classical
pathway, which is hypothalamic-pituitary-adrenal (HPA) axis, and whole
syndrome evolves in time through the three stages: (1) the alarm reaction, (2) the
stage of resistance, and (3) the stage of exhaustion. Each stage is indicated by
mobilization of the anterior pituitary-adrenal axis, involution of the thymio
lymphatic system, and the appearance of peptic ulcer, accordingly (Selye, 1976).
With 'fight and flight response' and GAS, it becomes possible to describe
the state of stress in physiological terms, and to approach the underlying
mechanism experimentally. These changes in biological concept of stress have
become the bases for a vast number of studies of stress in human beings and
animals which including poultry. Bayle (1967), as cited by Siegel (1995),
indicated that stress hormone in birds is primarily corticosterone. Ever since,
7
Recommended